Humoral Immune Responses Detailed Notes

Humoral Immune Responses

Phases and Types of Humoral Immune Responses

• Humoral immunity is mediated by antibodies, neutralizing and eliminating extracellular microbes and toxins.
• It's the main defense against microbes with polysaccharide and lipid-rich capsules.
• B cells produce antibodies for diverse extracellular molecules (polysaccharides, lipids, proteins).
• T cells recognize only protein antigens within cells.
• Naive B lymphocytes recognize antigens but don't secrete antibodies until activated.
• Activation leads to differentiation into antibody-secreting plasma cells.
• Key questions:
  • How are naive B cells activated and converted to antibody-secreting cells?
  • How is B cell activation regulated for different microbes?
• Naive B cells express membrane-bound IgM and IgD as antigen receptors.
• Activation of B lymphocytes:
  • Antigen-binding to membrane Ig.
  • Other signals (discussed later).
• Activation results in:
  • Clonal expansion (proliferation of antigen-specific cells).
  • Differentiation into plasma cells (antibody secretion).
• Antibodies secreted have the same specificity as the surface receptors on the B cells that initiated the response.
• One activated B cell can generate up to 4000 plasma cells, each producing up to $10^{12}$ antibody molecules per day.
• Heavy-chain isotype (class) switching:
  • B cells produce antibodies of different heavy-chain isotypes.
  • Mediates different effector functions against different microbes.
• Affinity maturation:
  • Repeated exposure to protein antigen increases antibody affinity.
  • Improves binding and neutralization of microbes/toxins.
• Antibody responses are classified as T-dependent or T-independent, based on the need for T cell help.
• B cells recognize diverse antigens (proteins, polysaccharides, lipids, nucleic acids, chemicals).
• Protein antigens are processed by APCs and recognized by helper T lymphocytes, which induce isotype switching and affinity maturation.
• Protein antigens elicit weak/no antibody responses without T cell help; thus, they are T-dependent.
• Nonprotein antigens (polysaccharides, lipids) stimulate antibody production without T cell involvement; thus, they are T-independent.
• T-independent responses show limited isotype switching and affinity maturation.
• Effective antibody responses require helper T cells.
• Follicular B cells:
  • Located in lymphoid organ follicles.
  • Make T-dependent, class-switched, high-affinity antibody responses to protein antigens.
  • Give rise to long-lived plasma cells.
• Marginal-zone B cells:
  • Located in splenic white pulp.
  • Respond to blood-borne polysaccharide antigens.
• B-1 cells:
  • Respond to nonprotein antigens in mucosal tissues and peritoneum.
• Marginal-zone B cells and B-1 cells:
  • Express antigen receptors of limited diversity.
  • Make predominantly IgM responses.
  • Lack features of T-dependent responses.
• Response types are not absolute; follicular B cells can make T-independent responses, and marginal-zone B cells can make some T-dependent responses.
• Primary and secondary antibody responses differ quantitatively and qualitatively.
• Primary response:
  • Smaller amounts of antibody produced.
• Secondary response:
  • Larger amounts of antibodies produced.
  • Increased heavy-chain isotype switching and affinity maturation.
  • Repeated stimulation increases helper T lymphocyte number and activity.
• These secondary response features are mainly seen with protein antigens, as T cells need to be activated, and only proteins activate T cells.

Stimulation of B Lymphocytes by Antigen

• Humoral responses start when antigen-specific B cells in spleen, lymph nodes, and mucosal lymphoid tissues recognize antigens.
• Antigens are transported to and concentrated in B cell–rich follicles and marginal zones of peripheral lymphoid organs.
• Macrophages lining the subcapsular sinus in lymph nodes capture antigens and display them to B cells in adjacent follicles.
• B cells use membrane-bound Ig receptors to directly recognize native (unprocessed) antigens.
• Antibodies secreted can bind to the native microbe or microbial product.
• Antigen recognition triggers signaling pathways that initiate B cell activation.
• B cell activation requires additional signals from innate immune reactions to microbes.

Antigen-Induced Signaling in B Cells

• Antigen-induced clustering of membrane Ig receptors triggers biochemical signals transduced by receptor-associated signaling molecules.

• B lymphocyte activation is similar to T cell activation.

• Ig receptor–mediated signal transduction requires cross-linking of two or more receptor molecules.

• Cross-linking occurs when:

  • Two or more antigen molecules in an aggregate bind to adjacent membrane Ig molecules.
  • Repeating epitopes of one antigen molecule bind to adjacent membrane Ig molecules.

• Polysaccharides, lipids, and nonprotein antigens often contain identical epitopes and can bind to numerous Ig receptors simultaneously.

• Protein antigens may be expressed in an array on the surface of microbes, enabling cross-linking of multiple antigen receptors on a B cell.

• Membrane IgM and IgD have short cytoplasmic tails and do not transduce signals themselves.

• The receptors are noncovalently associated with two proteins, called Igα and Igβ, to form the B cell receptor (BCR) complex, analogous to the T cell receptor (TCR) complex of T lymphocytes.

• The cytoplasmic domains of Igα and Igβ contain conserved immunoreceptor tyrosine-based activation motifs (ITAMs).

• When two or more antigen receptors of a B cell are clustered, the tyrosines in the ITAMs of Igα and Igβ are phosphorylated by kinases associated with the BCR complex.

• These phosphotyrosines recruit the Syk tyrosine kinase (equivalent to ZAP-70 in T cells), which is activated and phosphorylates tyrosine residues on adaptor proteins to recruit downstream signaling molecules.

• The net result of receptor-induced signaling in B cells is the activation of transcription factors that switch on the expression of genes whose protein products are involved in B cell proliferation and differentiation.

Role of Complement Proteins and Other Innate Immune Signals in B Cell Activation

• B lymphocytes express a receptor for a protein of the complement system that provides signals for their activation.
• The complement system is activated by microbes and by antibodies attached to microbes and functions as effector mechanisms of host defense.
• When the complement system is activated by a microbe, the microbe becomes coated with proteolytic fragments of the most abundant complement protein, C3.
• One of these fragments is called C3d.
• B lymphocytes express complement receptor type 2 (CR2, or CD21), which binds C3d.
• B cells that are specific for a microbe's antigens recognize the antigen by their Ig receptors and simultaneously recognize the bound C3d via the CR2 receptor.
• Engagement of CR2 enhances antigen-dependent activation responses of B cells.
• This illustrates that microbes or innate immune responses to microbes provide signals in addition to antigen that are necessary for lymphocyte activation.
• Complement activation represents one way in which innate immunity facilitates B lymphocyte activation, similar in principle to the role of costimulators of APCs for T lymphocytes.
• B lymphocytes express numerous Toll-like receptors (TLRs).
• TLR engagement on B cells by microbial products triggers activating signals that work with signals from the antigen receptor.
• This combination of signals results in optimal B cell proliferation, differentiation, and Ig secretion, promoting antibody responses against microbes.

Functional Consequences of B Cell Activation by Antigen

• B cell activation by antigen (and other signals) initiates proliferation and differentiation and prepares them to interact with helper T lymphocytes if the antigen is a protein.
• Activated B cells enter the cell cycle and begin to proliferate.
• The cells may also begin to synthesize more IgM and produce some of this IgM in a secreted form.
• Antigen stimulation induces the early phase of the humoral immune response.
• This response is greatest when the antigen is multivalent, cross-links many antigen receptors, and activates complement strongly.
• Most soluble protein antigens do not contain multiple identical epitopes, cannot cross-link many receptors on B cells, and do not stimulate high levels of B cell proliferation and differentiation by themselves.
• Protein antigens can induce signals in B lymphocytes that lead to important changes in the cells that enhance their ability to interact with helper T lymphocytes.
• Activated B cells endocytose protein antigen that binds specifically to the B cell receptor, resulting in degradation of the antigen and display of peptides in a form that can be recognized by helper T cells.
• Activated B cells reduce their expression of receptors for chemokines that are produced in lymphoid follicles and whose function is to keep the B cells in these follicles.
• At the same time, there is increased expression of receptors for chemokines that are produced in the T cell zones of lymphoid organs.
• As a result, the activated B cells migrate out of the follicles and toward the anatomic compartment where helper T cells are concentrated.
• Because of these changes, the B cells are poised to interact with and respond to helper T cells that have been activated by the same antigen presented to naive T cells by dendritic cells.
• Antibody responses to protein antigens require the participation of helper T cells.

Function of Helper T Lymphocytes in Humoral Immune Responses to Protein Antigens

• For a protein antigen to stimulate an antibody response, B lymphocytes and helper T lymphocytes specific for that antigen must come together in lymphoid organs and interact in a way that stimulates B cell proliferation and differentiation.

• Protein antigens elicit excellent antibody responses within 3 to 7 days after antigen exposure.

• The process of T-B cell interaction and T cell–dependent antibody responses occurs in a series of sequential steps:

  1. CD4 helper T cells and B cells are independently activated by a protein antigen in different regions of a lymphoid organ and migrate toward each other.
  2. These T and B cells initially interact outside the follicles.
  3. The initial antigen-specific T-B cell interaction consists of two phases:
     • B cells process and present antigen to the T cells.
     • The previously activated helper T cells express CD40 ligand and secrete cytokines, which act on the B cells to initiate proliferation and differentiation to plasma cells.
  4. Some activated B cells migrate back into the follicle, accompanied by helper T cells that were further activated by the B lymphocytes to develop into follicular helper T cells (T cells).
     • In response to signals from the T cells, B cells begin to proliferate, forming an organized structure called a germinal center, and the proliferating germinal center B cells undergo extensive somatic mutation of antibody gene variable regions and Ig heavy-chain isotype switching.
     • High-affinity B cells are selected in the germinal center, resulting in the production of high-affinity antibodies.
     • This germinal center reaction also results in the generation of long-lived plasma cells (many of which migrate to the bone marrow) and memory B cells.
       Activation and Migration of Helper T Cells

• Helper T cells that have been activated by dendritic cells migrate toward the B cell zone and interact with antigen-stimulated B lymphocytes in parafollicular areas of the peripheral lymphoid organs.

• The initial activation of T cells requires antigen recognition and costimulation.

• +The antigens that stimulate CD4 helper T cells are proteins typically derived from extracellular microbes that are internalized, processed in late endosomes and lysosomes, and displayed bound to class II major histocompatibility complex (MHC) molecules of APCs in the T cell–rich zones of peripheral lymphoid tissues.

• CD4+ T cells differentiate into effector cells capable of producing various cytokines, and some of these T lymphocytes migrate toward the edges of lymphoid follicles as antigen-stimulated B lymphocytes within the follicles are beginning to migrate outward.

• The directed migration of activated B and T cells toward one another depends on changes in the expression of certain chemokine receptors on the activated lymphocytes.

• On activation, T cells reduce expression of the chemokine receptor CCR7, which recognizes chemokines produced in T cell zones, and increase expression of the chemokine receptor CXCR5, which promotes migration into B cell follicles.

• B cells, on activation, undergo precisely the opposite changes, decreasing CXCR5 and increasing CCR7 expression.

• As a result, antigen-activated B and T cells migrate toward one another and meet at the edges of lymphoid follicles or in interfollicular areas.

Presentation of Antigens by B Lymphocytes to Helper T Cells

• The B lymphocytes that bind protein antigens by their membrane Ig antigen receptors endocytose these antigens, process them in endosomal vesicles, and display class II MHC–associated peptides for recognition by CD4 helper T cells.
• The membrane Ig of B cells is a high-affinity receptor that enables a B cell to specifically bind a particular antigen, even when the extracellular concentration of the antigen is very low.
• In addition, antigen bound by membrane Ig is endocytosed efficiently and is delivered to late endosomal vesicles and lysosomes, where proteins are processed into peptides that bind to class II MHC molecules.
• B lymphocytes are efficient APCs for the antigens they specifically recognize.
• B cells and T cells recognize different epitopes of the same protein antigen.
• B cells are capable of activating previously differentiated effector T cells but are inefficient at initiating the responses of naive T cells.
• A hapten is a small chemical that is recognized by B cells but stimulates strong antibody responses only if it is attached to a carrier protein.
• Some bacteria have polysaccharide-rich capsules, and the polysaccharides themselves stimulate weak (T-independent) antibody responses, especially in infants and young children. If the polysaccharide is coupled to a carrier protein, however, effective responses are induced against the polysaccharide because helper T cells are engaged in the response.
• Such conjugate vaccines have been very useful for inducing protective immunity against bacteria such as Haemophilus influenzae, especially in infants.

Mechanisms of Helper T Cell–Mediated Activation of B Lymphocytes

• Helper T lymphocytes that recognize antigen presented by B cells express CD40 ligand (CD40L) and secrete cytokines, which activate the antigen-specific B cells.
• The process of helper T cell–mediated B lymphocyte activation is analogous to the process of T cell–mediated macrophage activation in cell-mediated immunity.
• CD40L expressed on activated helper T cells binds to CD40 on B lymphocytes. Engagement of CD40 delivers signals to the B cells that stimulate proliferation (clonal expansion) and the synthesis and secretion of antibodies.
• At the same time, cytokines produced by the helper T cells bind to cytokine receptors on B lymphocytes and stimulate more B cell proliferation and Ig production.
• The requirement for the CD40L-CD40 interaction ensures that only T and B lymphocytes in physical contact engage in productive interactions.
• Helper T cell signals also stimulate heavy-chain isotype switching and affinity maturation, which typically are seen in antibody responses to T-dependent protein antigens.

Extrafollicular and Germinal Center Reactions

• The initial T-B interaction, which occurs at the edge of lymphoid follicles, results in the production of low levels of antibodies, which may be of switched isotypes but generally of low affinity.
• The plasma cells that are generated in this reaction are typically short-lived and produce antibodies for a few weeks, and few memory B cells are generated.
• Some of the helper T cells that are activated by B lymphocytes express high levels of the chemokine receptor CXCR5, which draws these T cells into the adjacent follicles.
• The CD4 T cells that migrate into B cell–rich follicles are called follicular helper T (T ) cells. The generation and function of T cells are dependent on a costimulator of the CD28 family called ICOS (inducible costimulator).
• T cells may develop from uncommitted T cells or from other subsets, including T 1, T 2, and T 17, and may also secrete cytokines, such as IFN-γ, IL-4, or IL-17, which are characteristic of these subsets.
• Most T cells also secrete the cytokine IL-21, whose role in antibody production is a topic of active research.
• A few of the activated B cells from the extrafollicular focus migrate back into the lymphoid follicle and begin to divide rapidly in response to signals from T cells. It is estimated that these B cells have a doubling time of approximately 6 hours, so that one cell may produce about 5000 progeny within a week.
• The region of the follicle containing these proliferating B cells is the germinal center.
• Germinal center B cells undergo extensive isotype switching and somatic mutation of Ig genes.
• The highest affinity B cells are the ones that are selected at the end of the germinal center reaction to differentiate into memory B cells and long-lived plasma cells.

Heavy-Chain Isotype (Class) Switching

• Helper T cells stimulate the progeny of IgM and IgD–expressing B lymphocytes to produce antibodies of different heavy-chain isotypes (classes).

• Different antibody isotypes perform different functions, and therefore the process of isotype switching broadens the functional capabilities of humoral immune responses.

• Effective host defense requires that the immune system make different antibody isotypes in response to different types of microbes, even though all naive B lymphocytes specific for all these microbes express antigen receptors of the IgM and IgD isotypes.

• Another functional consequence of isotype switching is that IgG antibodies produced are able to bind to a specialized Fc receptor called the neonatal Fc receptor (FcRn).

• Heavy-chain isotype switching is induced by a combination of CD40L-mediated signals and cytokines. These signals act on antigen-stimulated B cells and induce switching in some of the progeny of these cells.

• In the absence of CD40 or CD40L, B cells secrete only IgM and fail to switch to other isotypes, indicating the essential role of this ligand- receptor pair in class switching.

• A disease called the X-linked hyper-IgM syndrome is caused by mutations in the CD40L gene, which is located on the X chromosome, leading to production of nonfunctional forms of CD40L. In this disease, much of the serum antibody is IgM, because of defective heavy-chain class switching.

• Molecular basis of heavy-chain isotype switching:

  • IgM-producing B cells contain a rearranged VDJ gene adjacent to the first constant-region cluster, which is Cµ.
  • The heavy-chain mRNA is produced by splicing a VDJ exon to Cµ exons in the initially transcribed RNA, and this mRNA is translated to produce µ heavy chain, which combines with a light chain to give rise to IgM antibody.
  • Signals from CD40 and cytokine receptors stimulate transcription through one of the constant regions that is downstream of Cµ. In the intron 5′ of each constant region (except Cδ) is a conserved nucleotide sequence called the switch region. When a downstream constant region becomes transcriptionally active, the switch region 5′ of Cµ recombines with the switch region 5′ of that downstream constant region, and the intervening DNA is deleted.
  • An enzyme called activation-induced deaminase (AID), which is induced by CD40 signals, plays a key role in this process.
  • AID converts cytosines (C) in DNA to uracil (U). The sequential action of other enzymes results in the removal of the U’s and the creation of nicks in the DNA. Such a process on both strands leads to double-stranded DNA breaks When double-stranded DNA breaks in two switch regions are brought together and repaired, the intervening DNA is deleted, and a rearranged VDJ exon that was originally close to Cµ may now be brought immediately upstream of the constant region of a different isotype (e.g., IgG, IgA, IgE).
  • This process is called switch recombination. The result is that the B cell begins to produce a new heavy-chain isotype (determined by the C region of the antibody) with the same specificity as that of the original B cell, because specificity is determined by the rearranged VDJ exon.
  • Cytokines produced by helper T cells determine which heavy-chain isotype is produced by influencing which heavy-chain constant-region gene participates in switch recombination.

Affinity Maturation

• Affinity maturation is the process by which the affinity of antibodies produced in response to a protein antigen increases with prolonged or repeated exposure to that antigen.
• This increase in affinity is caused by point mutations in the V regions, and particularly in the antigen-binding hypervariable regions, of the genes encoding the antibodies produced.
• Affinity maturation is seen only in responses to helper T cell–dependent protein antigens, indicating that helper cells are critical in the process.
• Affinity maturation occurs in the germinal centers of lymphoid follicles and is the result of somatic hypermutation of Ig genes in dividing B cells, followed by the selection of high-affinity B cells by antigen.
• In germinal centers, the Ig genes of rapidly dividing B cells undergo numerous point mutations. The enzyme AID required for isotype switching also plays a critical role in somatic mutation. The U's that are produced by this enzyme in Ig V-region DNA are frequently converted to T's during DNA replication, or they are removed and repaired by error- prone mechanisms that often lead to mutations. The frequency of Ig gene mutations is estimated to be one in $10^3$ base pairs per cell per division, about 100,000-fold to 1 million–fold greater than the mutation rate in most other genes. For this reason, Ig mutation in germinal center B cells is called somatic hypermutation.
• This extensive mutation results in the generation of different B cell clones whose Ig molecules may bind with widely varying affinities to the antigen that initiated the response.
• Germinal center B cells undergo apoptosis unless rescued by antigen recognition and T cell help.
• High-affinity B cells most efficiently bind the antigen and are activated to survive. These B cells also internalize the antigen, process it, and present peptides to germinal center T cells, which then provide critical survival signals.
• Antibody-secreting cells that are produced in the germinal centers have also been called plasmablasts because they are not fully differentiated.
• These cells enter the circulation and tend to migrate to the bone marrow, where they mature into plasma cells and may survive for years and continue to produce high-affinity antibodies, even after the antigen is eliminated.
• A fraction of the activated B cells, which often are the progeny of isotype-switched high-affinity B cells, do not differentiate into active antibody secretors but instead become memory cells.
• Memory B cells do not secrete antibodies, but they circulate in the blood and reside in various tissues. They survive for months or years in the absence of additional antigen exposure, ready to respond rapidly if the antigen is reintroduced.

Antibody Responses to T-Independent Antigens

• Polysaccharides, lipids, and other nonprotein antigens elicit antibody responses without the participation of helper T cells.
• These nonprotein antigens cannot bind to MHC molecules, so they cannot be seen by T cells.
• Antibody responses to T- independent antigens differ from responses to proteins, and most of these differences are attributable to the roles of helper T cells in antibody responses to proteins.
• Because polysaccharide and lipid antigens often contain multivalent arrays of the same epitope, these antigens may be able to cross-link many antigen receptors on a specific B cell. This extensive cross-linking may activate the B cells strongly enough to stimulate their proliferation and differentiation without a requirement for T cell help.

Regulation of Humoral Immune Responses: Antibody Feedback

• After B lymphocytes differentiate into antibody-secreting cells and memory cells, a fraction of these cells survive for long periods, but most of the activated B cells probably die by a process of programmed cell death. This gradual loss of the activated B cells contributes to the physiologic decline of the humoral immune response.
• As IgG antibody is produced and circulates throughout the body, the antibody binds to antigen that is still available in the blood and tissues, forming immune complexes. B cells specific for the antigen may bind the antigen part of the immune complex by their Ig receptors. At the same time, the Fc tail of the attached IgG antibody may be recognized by a special type of Fc receptor expressed on B cells (as well as on many myeloid cells) called FcγRIIB
• This Fc receptor delivers inhibitory signals that shut off antigen receptor–induced signals, thereby terminating B cell responses. This process, in which antibody bound to antigen inhibits further antibody production, is called antibody feedback. It serves to terminate humoral immune responses once sufficient quantities of IgG antibodies have been produced.